KR101029306B1 - Method for determination of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid - Google Patents

Abstract

PURPOSE: A method for analyzing 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid is provided to overcome an emulsion phenomenon and reduce the amount of a solvent required for an extracting process. CONSTITUTION: A hair sample is introduced into a polypropylene tube containing stainless steel beads and is automatically fine-cut. The fine-cut sample is hydrolyzes using a strong basic solution. A solvent is added to the hydrolyzed sample. A liquid phase is extracted from the sample using beads. A super-rapid centrifugal process is implemented. The extracted solution is undergone a derivatization chemical reaction. The derived solution is separated using a gas chromatography.

Description

Method for determination of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid}

The present invention is a method for analyzing hemp metabolites in hair, and more particularly, an analysis method including gas chromatograph through liquid phase extraction, centrifugation, and derivatization using beads.

Hemp (cannabis) is the most abusive drug in the world, and it is the second highest drug abuse after amphetamine drugs in Korea. The active substance of hemp is tetrahydrocannabinol (delta 9-tetrahydrocannabinol (THC)). When hemp is smoked, he undergoes metabolic processes in the body, and then 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid. , THC-COOH).

In general, the parent drug and metabolite or one component of the metabolite may be analyzed to determine whether the drug is taken. Therefore, to determine whether or not smoking hemp, THC-COOH, a metabolite produced in the body's metabolic process, should be analyzed.

Typically, the samples used for drug analysis are urine and hair. Hemp can be checked for 1 ~ 2 weeks after smoking due to urine sensation, while hair emotion can be estimated not only for smoking several months ago but also for abuse history such as timing and frequency.

However, to confirm whether hemp was used illegally until recently, only urine analysis could be used to confirm whether it was taken. This is because, unlike other drugs, the rate at which hemp metabolite metastases are significantly lowered to the hair, resulting in a very low concentration of residual hemp in the hair, making it difficult to analyze.

Therefore, in order to perform effective hemp hair analysis, it is essential to introduce a high sensitivity analysis method that can analyze trace amounts, and due to this difficulty, only a few countries, such as the United States and Germany, have applied the hair emotion technique for the hemp metabolites to actual emotion until recently. It is a situation where it is developing its own development.

At present, the drug market in Korea is not only diversified in importing countries, but also the international distribution of drugs through foreign workers, overseas Koreans, and international students is more frequent than in the past, making it easy to bring drugs into Korea. Influx of hemp is much more hallucinogenic than conventional cannabis, and various types of drugs, including hemp ingredients, such as hemp cookies, which are easy to store and take, are distributed, and the composition ratio of hemp criminals is increasing every year. As the frequency becomes more frequent, the demands and interests on the development and application of the hemp hair analysis method, which can confirm the long-term use status, are focused.

As described above, the hair analysis allows the drug to accumulate and grow in the hair from the hair root, so it is possible to check whether the drug is taken for the length corresponding to the length of the hair, and to check whether or not to take the habitual hair through the hair sectional analysis. There is an advantage. On the other hand, the analytical process is more complicated than urine analysis, requires a lot of human labor, and requires a trace amount analysis.

The general hemp metabolite hair analysis process can be divided into six steps. Decontamination, pulverization, incubation, extraction, purification and instrumental analysis of the hair. A series of hair analysis processes is generally considered an analytical method that takes a long time to process and requires considerable labor.

The analysis of hemp metabolites in conventional hair is a labor intensive and long processing time. Therefore, it is necessary to shorten the time at the hair cutting stage, and efforts have been made to reduce the time and stage required at the extraction stage.

The liquid phase extraction method for extracting hemp metabolites using organic solvents from a conventional thin sample is to extract the analyte by shaking with a sample and a solvent in a test tube. There was a problem of lowering the extraction rate of the analyte.

Therefore, in order to increase the extraction rate of hemp metabolites, a new method that can be extracted without being affected by the emulsion is required.

After the extraction step, the purification process is a step for effectively removing a large amount of degradation products of the hair protein generated during the hydrolysis process. These interferences are purified through the centrifugation process after liquid phase extraction.

According to the conventional liquid phase extraction method, the test tube containing the sample subjected to the extraction process tends to be damaged due to damage to the test tube when centrifugation at 3200 g or more, so that sufficient layer separation cannot be achieved at high speed, resulting in clean purification of the extract. You may not. High speed centrifugation is a method that can be centrifuged at high speed and undergo a desired purification process. This method can use the same crushing container used in the automatic washing method can minimize the loss of the sample, and proceed to ensure effective purification in a short time.

The analyzer used as the standard for analyzing drugs in biological samples is a gas chromatograph-mass spectrometer (GC-MS). First, the analyte is separated by chromatography, and then the mass is identified. This has a possible advantage. Therefore, the method used to check whether or not to take drugs in the hair is also frequently used a combination of chromatography and mass spectrometry.

Recently, liquid chromatograph-mass spectrometers have been reported for the analysis of various drugs in materials that are difficult to separate into gas chromatographs or materials with low thermal stability. However, when analyzing polar materials using gas chromatography-mass spectrometry, various derivatization methods for converting polar functional groups of analytes into nonpolar are commercially available, and thus, polar materials can be analyzed using gas chromatography.

The derivatization method used for THC-COOH analysis as a conventional technique is based on various amphetamine-based stimulants by applying trimethylsilylation method using N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) derivatization reagent and electron impact ionization method. Although there have been applications for simultaneous analysis of components, this method is not suitable for trace analysis using chemical ionization because it does not contain atoms containing electrons such as florin.

One conventional method incorporates an additional purification step to remove analyte that is excessively extracted from the hair substrate (Sachs and Dressler, Forensic Sci Int., 107 (2000) 239). These methods involve additional steps that eventually complicate the preprocessing process and make it difficult to use for routine analysis.

Therefore, in order to establish a method for rapid and cost-effective economic and high sensitivity analysis of hemp metabolites in hair, the pretreatment step is automated and simplified to be suitable for rapid analysis, and a small amount of solvent is used to extract analytes. It includes a purification step that facilitates and effectively removes interfering substances, and it is required to develop a drug analysis method that can obtain sufficient analytical sensitivity by applying a derivatization chemical reaction and an appropriate ionization method and can be easily analyzed on a daily basis. It is true.

It can overcome the emulsion phenomenon, which is the main factor that lowers the recovery rate during the extraction process, reduce the amount of solvent used for extraction, and use high speed centrifugation method which is effective for removing interfering substances and sample purification and derivatization method suitable for chemical ionization. Through this purpose, high-sensitivity analysis of hemp metabolites is possible.

However, technical problems to be achieved by the present invention are not limited to the above-mentioned problems, and other technical problems will be clearly understood by the average technician from the following description.

In order to achieve the above object, in one embodiment of the present invention, the step of cutting the hair sample to a particle size of millimeter level; Hydrolyzing the sample obtained in the cutting step into a strong basic solution; Centrifuging the sample obtained in the hydrolysis step after extracting the liquid phase using beads; And it provides a hemp metabolite analysis method comprising the step of separating the extract after the centrifugation step using a derivative compound through gas chromatography.

In another embodiment of the present invention, there is provided a hemp metabolite analysis method for further analyzing the compound separated by the gas chromatograph by a mass spectrometer through a process of negative ion chemical ionization (negative ion chemical ionization).

In another embodiment of the invention, the hemp metabolite is tetrahydrocannabinol carboxylic acid (THC-COOH).

In another embodiment of the present invention, the step of slicing the sample is placed in a 1.5-2 mL volume tube containing 2.8-3.0 mm diameter stainless steel beads (stainless steel bead) for 8-10 minutes at 30 ± 5 Hz While auto trimming.

In another embodiment of the present invention, the step of extracting the liquid phase using the beads is 10 after adding a mixed solvent n-hexane: ethyl acetate (9: 1, v / v) to the sample obtained in the hydrolysis step Extract at ± 2 Hz.

In another embodiment of the present invention, the centrifugation in the step of centrifugation after the liquid phase extraction is performed at 10000 ~ 30000 g, 3 to 5 minutes.

In another embodiment of the present invention, the derivative compound is pentafluoropropionic anhydride (PFPA) / pentafluoropropanol (PFPOH).

In another embodiment of the present invention, the mass spectrometer uses a tandem mass spectrometer.

Other specific details of embodiments of the present invention are included in the following detailed description.

According to the present invention, dozens of hair samples can be automatically cut at the same time in one cut to shorten the pretreatment time, and the liquid phase extraction using beads has been newly devised to solve the phenomenon of emulsion, which is a major factor in the reduction of the hemp metabolite extraction rate. Removal significantly improved extraction efficiency and significantly reduced the amount of solvent used.

In addition, by extracting the liquid phase using the beads, it is possible to increase the efficiency of separation by introducing a high-speed centrifugation method in the purification process, high sensitivity analysis by introducing a chemical ionization method and a suitable derivatization process.

FIG. 1 is a chromatogram showing the results of analysis using a gas chromatograph-dual mass spectrometer applied to anion chemical ionization using hair of a real hemp smoker.
Figure 2 is a graph showing the concentration distribution of hemp metabolites measured in the hair of a real hemp smoker (224 people) according to the present invention.

Hereinafter, embodiments of the present invention will be described in detail. However, this is presented as an example, by which the present invention is not limited, and the present invention is defined only by the scope of the claims to be described later.

According to one aspect of the invention, the step of cutting the hair sample to the particle size of the millimeter level; Hydrolyzing the sample obtained in the cutting step into a strong basic solution; Centrifuging the sample obtained in the hydrolysis step after extracting the liquid phase using beads; And it provides a hemp metabolite analysis method comprising the step of separating the extract after the centrifugation step using a derivative compound through gas chromatography.

The hemp metabolite is tetrahydrocannabinol carboxylic acid (THC-COOH), and is represented by the following Chemical Formula 1.

[Formula 1]

The hemp metabolite is a biological metabolite of tetrahydrocannabinol (THC), and THC is represented by the following Chemical Formula 2.

[Formula 2]

Hemp Metabolite Tetrahydrocannabinol carboxylic acid is produced by metabolic process of tetrahydrocannabinol (THC), a major active ingredient related to hallucination among cannabinoids contained in hemp. Currently, THC-COOH is used as a reference material to determine whether to smoke hemp in urine and hair analysis. In general, THC-COOH in urine is known to be available for 4-8 hours after hemp smoking and up to 4 weeks. Depending on the length of hair, you can check for smoking within a few months to a year.

In the analytical method of the present invention, the hair sample cutting step cuts the sample to a size below the millimeter level so that the proteins constituting the hair are easily hydrolyzed in the basic solution.

Hair cutting method is not particularly limited, but according to one embodiment of the present invention, it is preferable to use an automatic grinding device that can quickly process a large amount of samples and can reduce labor costs. According to one embodiment of the invention, the sample is preferably placed in a 1.5-2 mL volume tube containing 2.8-3.0 mm diameter stainless steel beads and automatically washed for 8-10 minutes at 30 ± 5 Hz. Bow down.

The hydrolysis process can be performed to effectively dissociate the hemp metabolite component bound to the hair protein, and the applicable method for the hydrolysis is an enzyme-based method, a strong base solution (1-2 M aqueous sodium hydroxide solution). The method of using, the method of using ultrasonic waves, etc. may be used, but according to one embodiment of the present invention, it is preferably hydrolyzed using an aqueous sodium hydroxide solution.

In the present invention, the sample obtained in the hydrolysis step comprises the step of centrifugation after liquid phase extraction using beads.

Usually, in the step of extracting the sample obtained in the hydrolysis step, liquid extraction method, solid phase extraction method and direct extraction by solvent under ultrasonic method are used as extraction methods.

In the conventional liquid phase extraction method for extracting analyte from an aqueous layer into an organic layer, an analyte is generated when agitation speed is increased by adding a sample and a solvent to a test tube and shaking the sample. There is a problem of reducing the extraction efficiency of the analyte. Therefore, in order to increase the extraction rate, it is possible to improve the extraction rate by lowering the shaking speed and increasing the shaking time, but there is a disadvantage that the overall extraction time increases.

In addition, in the liquid phase extraction method, the sample contained in the test tube is extracted with a shaker by putting an organic solvent. The amount of the organic solvent used for extraction is about 3 to 5 mL.

In the case of the test tube used for the extraction, since the volume of the sample and the solvent is large, it is not possible to use a test tube capable of high-speed centrifugation, and thus a test tube made of a candle is usually used, so it is usually centrifuged at 3200 g or more to separate the aqueous layer and the organic layer. If you do, the test tube is damaged and tends to break. Thus, sufficient layer separation at high speeds may not be achieved and eventually no clean purification of the extract may result.

Liquid phase extraction using the beads of the present invention can increase the extraction efficiency without being affected by the emulsion phenomenon, after which the high-speed centrifugation is possible during centrifugation, it can be reduced by more than 10 times than the conventional centrifugation time. For example, according to one embodiment of the present invention, the extraction time of about 20 minutes can be shortened to 2 minutes, which is a level of 10/10, and in particular, a high-strength container can be adopted to withstand high-speed rotational force of up to 30000 g during centrifugation. .

Liquid phase extraction using the bead of the present invention is extracted by adding a small amount of organic solvent to a small amount of sample in the state that the stainless steel beads (2.8 mm-3.0 mm in diameter) used in the cutting is added to a 2 mL tube made of polypropylene This is going on. According to one embodiment of the invention, it is possible to extract by adding 0.8 mL of organic solvent to 0.7 mL of sample. At this time, the role of the bead is to shake the water layer and the added organic solvent at a rate of 5-20 Hz by stirring to stir, so that sufficient extraction efficiency can be obtained in a short time. According to one embodiment of the invention, the liquid phase extraction is preferably extracted at 10 ± 2 Hz, and according to one embodiment of the invention, the extraction time is preferably at least 2 minutes.

According to one embodiment of the invention the solvent used for liquid phase extraction is preferably a mixture of normal nucleic acid and ethyl acetate, most preferably the ratio of the nucleic acid: ethyl acetate is 9: 1 (v / v).

Liquid phase extraction using beads can reduce the amount of solvent used by 1/3 compared to the conventional method, and the time of extracting a large amount of samples at a time significantly reduces the pretreatment time than the conventional method. Can be.

In addition, unlike a test tube made of a choja, because the sample and the extraction solvent is used in a small amount, it is possible to use a high-strength test tube capable of high-speed centrifugal separation in addition to the glass choja, there is an advantage that the high-speed centrifugal separation can separate the water layer and the organic layer cleanly.

The influence of the disturbances due to the matrix effect of the hair generated during the hydrolysis process is that the ions to be quantitated are detected less than the actual amount due to the substrate of the hair itself or vice versa. Therefore, it is necessary to reduce the influence of the substrate through the refining process of the extract and to effectively remove the interfering substances extracted with the analyte from the extract.

After the liquid phase extraction using the beads, in the step of centrifugation, according to one embodiment of the present invention, the extract can be purified by centrifugation at high speed centrifugation at 10000-30000 g for 3-5 minutes.

Through the above process, the substrate effect of the sample is reduced, so that the purified state is preferable, the reproducibility is high, and the quantitative analysis can be performed with a constant retention time.

After the centrifugation step, the extract is derivatized using a derivative compound. Derivative compounds are trifluoroacetic anhydride (TFAA), pentafluoropropionic anhydride (PFPA), heptaflourobutyric anhydride (HFBA), pentafluoropropanol (PFPOH) and hexafluropropanol (HFIP), and preferably pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH).

By using the derivatization technique, when the derivatization technique is not used, it is possible to effectively detect a compound that is difficult to analyze by adsorption of an analyte on a capillary column. When derivatization of hemp metabolites with PFPOH and PFPA using derivatization techniques, peak shape and intensity can be increased, improving linearity within the quantitative range, Provide improvement. At this time, the derivatization conditions can be set in the temperature range of 50 ~ 80 ℃, but preferably the reaction conditions of 30 minutes at 70 ℃ can be selected.

Purification of the re-dissolved extract is performed prior to instrumental analysis on the extract of which the derivative reaction is completed. In this case, as a preferred purification method, a syringe filter may be used to effectively filter out interferences. The filter is preferably made of polyvinylidene fluoride (PVDF) and has a particle size of 0.2 μm.

On the other hand, when the sample is analyzed using a gas chromatograph and a mass spectrometer, the components separated by the difference in adsorption or partition coefficient between the fixed and mobile phases of the capillary separation tube mounted on the gas chromatograph are different in retention time. Injected into the mass spectrometer. The introduced sample is ionized by the ionizer.

In the present invention, by using a capillary tube in the instrument analysis step, the analyte can be preferably detected. At this time, the capillary tube is composed of a 5% phenyl- and 95% methyl-polysiloxane stationary phase, it is preferable to use a capillary tube of 30 m in length, 0.25 mm in diameter, 0.25 μm in thickness of the stationary phase, When this is used, an increase in resolution and intensity can be achieved, providing improved detection sensitivity.

In the separation condition setting step, the temperature of the gas chromatograph's oven can be adjusted stepwise to adjust the retention time and separation condition of each analyte. The temperature conditions applied are then set to minimize the effects of interfering substances from the substrate and to allow all analytes to be analyzed in a short time.

In the temperature control step of the gas chromatograph, the isothermal and warming are applied to analyze the analyte, and the analyte and the interfering substance are separated, and the chromatogram of each component is improved in the temperature range of 100-300 ℃. Can be selected.

For gas chromatograph analysis, the analyte having a high molecular weight may select a flow rate of 0.7-1.4 mL / min in the capillary tube to remove peak tailing and improve sensitivity, preferably 1.0 mL / min. Can be used. Sample injection methods are split and splitless, but may be preferably used as a splitless method for high sensitivity analysis.

As a capillary separation tube for gas chromatograph, a separation tube having a diameter of 0.1-0.53 mm may be used. As the stationary phase of the separation tube, (5% -phenyl) -methylpolysiloxane and 100% dimethylpolysiloxane can be used. Nitrogen, hydrogen, helium gas may be used as the mobile phase, and preferably, high purity helium gas may be used.

Samples separated through a capillary tube of the gas chromatograph may be ionized using chemical ionization (NCI) prior to analysis by dual mass spectrometry (MS / MS). The ionization method may be an anion and a cationic chemical ionization method, preferably an anion chemical ionization method.

The ionized detection ion selects the characteristic ion from the mass spectrum obtained from the first quadrupole of the analyte, and the selected characteristic ion selects the characteristic ion from the mass spectrum obtained by passing through the third quadrupole. Select and analyze quantitative ion pairs and qualitative ion pairs.

In the step of analyzing by the dual mass spectrometer, the quantification in the multiple reaction monitoring (MRM) method of the mass spectrometer during qualitative and quantitative analysis is to improve the signal to noise ratio. In other words, it is preferable to select a characteristic ion pair of the analyte to be analyzed by MRM method detected by MS / MS.

In case of using the MRM method, two different ion pairs are selected for the analyte, and when the characteristic ion pairs of the deuterium-substituted internal standard are detected together, the selectivity is increased and the analysis sensitivity is improved. In addition, the analyte can be analyzed with high precision and accuracy.

Hereinafter, the Example and comparative example of this invention are described. The following examples are only preferred embodiments of the present invention, and the present invention is not limited to the following examples.

<Example>

One. Dozens  First step in automatically rinsing hair samples to sub-millimeter particle sizes

Cut hair washed with isopropyl alcohol to a size of 2 cm or less, take 25 mg of the sample and place it in a 2 mL tube containing 3 stainless steel beads 2.8 mm in diameter with a maximum throughput of 48 Germany The grinding machine (Qiagen Tissu Lyser II) was cut for 8 minutes at 30 Hz.

The size of the shredded hair particles was kept to a size of 1 millimeter or less, so as to be suitable for the hydrolysis reaction. Prior to hydrolysis, 50 μL of an internal standard solution (1.0 ng / mL for THC-COOH- d ₉ ) was added to the cut hair samples.

2. Fragmentation  Hydrolyze the sample with strong basic solution and add mixed organic solvent Bead  Used By liquid phase extraction  Extract, then By high speed centrifugation  Purifying and extracting the extract PFPA Of PFPOH Derivatization  By reaction With gas chromatograph  2nd step to separate

A hydrolysis reaction is carried out in the strong base solution that breaks the bond between the hair and the analyte. 0.7 mL of 1 M aqueous sodium hydroxide solution was added to the sample to which the internal standard was added, followed by heating at 95 ° C. for 30 minutes. Acidify by adding 120 μL of acetic acid to the fully hydrolyzed sample, add 0.8 mL of n-hexane: ethyl acetate (9: 1, v / v), and shake with beads for 2 minutes at 10 Hz. Was performed.

The extract was purified by high-speed centrifugation at 30000 g for 3 minutes. 30 μL of PFPOH and 50 μL of PFPA were added to the residue from which the purified extract was evaporated, and then a derivatization experiment was performed for 30 minutes in a reactor maintained at 70 ° C. At this time, the carboxylic acid group, which is the characteristic functional group of THC-COOH, was subjected to alkylation derivatization with PFPOH, and the acylation derivatization with alcohol was performed with PFPA. The mixture was evaporated under a nitrogen stream, and then 100 μL of ethyl acetate was added to the residue, and the mixed solution was purified by a syringe filter. As a syringe filter, a filter made of Whatman Teflon, UK, capable of passing particles of 0.2 μm or less was used.

3. Anion chemical ionization ( NCI Method and dual mass spectrometry MS Of MS Hemp in the hair using) Metabolites  Qualitative, quantitative third step

After syringe filtering in this step, a portion (1 μL) of the filtrate was taken and injected into a gas chromatograph-dual mass spectrometer. The gas chromatograph used was a gas chromatograph (6890N GC) manufactured by Agilent USA equipped with an autoinjector.

At this time, the temperature of the capillary tube was set to 100 ℃ and maintained for 1 minute, then heated to 35 ℃ / min to 275 ℃ and maintained for 3 minutes, then heated to 300 ℃ at 25 ℃ / min and maintained for 3.5 minutes. At this time, the temperature of the gas chromatograph inlet and the mass spectrometer was set to 260 and 280 ° C. The sample was injected in a splitless mode, and then, after 1 minute, the system was switched to the split mode.

As shown in Figure 1, when using the gas chromatograph of the present invention and the high sensitivity analysis of hemp metabolites in the hair using the dual mass spectrometer, it was possible to separate the analyte having different chemical properties in the step of passing through the capillary tube.

The dual mass spectrometer (MS / MS) used as a detector is a Quattro micro GC detector manufactured by US Waters, which ionizes the analyte by anion chemical ionization, using methane as a reactor. Collision gas used for MS / MS analysis was made of argon with a pressure of 3.0 ± 0.3 mTorr. The temperature of the ionizer and quadrupole were set at 230 and 150 ° C.

In addition, in order to improve the signal to noise ratio and to improve the detection sensitivity, the analysis was performed by the MRM method in which the analyte detects characteristic ions generated by the ionizer.

Table 1 shows the characteristic ion pairs of the analyte for qualitative and quantitative analysis. FIG. 1 shows an MRM chromatogram of a real hemp smoker detected by a dual mass spectrometer using an ion pair corresponding to an analyte and an internal standard.

As a result, the qualitative quantitative analysis of the gas chromatograph and detector of the present invention up to the detection limit of 0.015 pg / mg using the high sensitivity analysis method of hemp metabolite using a dual mass spectrometer was possible. In addition, the analysis time can be accelerated by automating the labor and time-consuming hair cutting process, and the liquid phase extraction method using the newly designed beads reduces the amount of solvent used, improves the extraction efficiency, and prevents interference from the substrate in the hair. The extract was purified more effectively by the high-speed centrifugation method, and shows that the industrial availability is great when the high sensitivity analysis method of the present invention is applied to the hair of a real hemp smoker (Table 3).

In the quantitative analysis, the area of the peaks shown in the MRM chromatogram was summed, and then an internal standard method was applied to draw a calibration curve to determine the content. The precision and accuracy of the assay were measured with low, medium and high concentration quality control samples (n = 6) and the results are shown in Table 2. In addition, the quantitative analysis results of the actual hemp smoker's hair sample are shown in FIG. 2.

TABLE 1

TABLE 2

[Table 3]

4. Extraction solvents of the present invention and the prior art and Fragmentation  Compare method

Liquid phase extraction method using the beads of the present invention and gas chromatography and chemical ionization method for the analysis of hemp metabolites in the hair cutting method and treatment capacity, extraction method, solvent usage, derivatization method, ionization method, detection limit (limit of detection, quantitative range values were compared with conventional hemp metabolite assays.

As a conventional technique, a method using a gas chromatograph-double mass spectrometer equipped with a chemical ionizer (Comparative Example 1: Marsili et al., Rapid Commun Mass Spectrom., 19 (2005) 1566), purification process using a liquid chromatograph The method of using a gas chromatograph-mass spectrometer equipped with a chemical ionizer (Comparative Example 2: Sachs and Dressler, Forensic Sci Int., 107 (2000) 239) was compared with the present invention. Is showing.

Example 1

Hemp metabolite analysis was performed by the first to third steps.

Comparative Example 1

Using a gas chromatograph-double mass spectrometer equipped with a chemical ionizer, the hair is cut with scissors and hydrolyzed with a strong base solution, followed by liquid phase extraction using a mixed solvent of heptane: ethyl acetate, followed by PFPA. Samples derivatized with / PFPOH were separated by capillary tubes and hemp metabolite analysis was performed.

Comparative Example 2

Purification by liquid chromatograph and gas chromatograph-mass spectrometer equipped with anionic chemical ionization device. Scissor hair using scissors, hydrolyze into strong base solution, and then use n-hexane: ethyl acetate mixed solvent. After performing liquid phase extraction by using the liquid chromatography, the sample was purified by liquid chromatography, and the sample was extracted again by liquid phase extraction with n-hexane: ethyl acetate mixed solvent. Hemp metabolite analysis was performed through the tube.

[Table 4]

Comparing the examples with the comparative examples, it is shown that the analytical method of the present invention is quite effective for the analysis of hemp metabolites. This process includes the automatic washing step of the sample, the liquid phase extraction method using beads, the purification step, the derivatization process suitable for the anion chemical ionization method (NCI) as the last step, and the instrumental analysis using the dual mass spectrometer (MS / MS) as the detector. When using the analysis method comprising the step has the advantages of high analysis speed and simplicity, cost reduction through automation, securing the desired analysis sensitivity, accuracy of the analysis results, reproducibility.

Claims (8)

A 20-25 mg hair sample is placed in a 1.5 to 2 mL polypropylene tube containing 2.8 to 3.0 mm stainless steel beads in diameter and milled to a particle size of 8 to 10 minutes at 30 ± 5 Hz. Automatic cutting step;
Hydrolyzing the sample obtained in the automatic cutting step into a strong basic solution;
Adding 0.5-2.0 mL of solvent to the sample obtained in the hydrolysis step and extracting the liquid phase using beads for 1-5 minutes, then performing ultrafast centrifugation at 10,000-30,000 g for 3 to 5 minutes; And
After the centrifugation step comprises the step of separating the extract by gas chromatography through derivatization using a derivative compound,
The automatic shredding, hydrolysis and ultrafast centrifugation after liquid phase extraction are performed in a 1.5-2 mL volume polypropylene tube used in the automatic shredding. Way. The method of claim 1, wherein the analysis method further comprises analyzing the compound separated by the gas chromatograph by a mass spectrometer through a negative ion chemical ionization process. . The method of claim 1, wherein the hemp metabolite is tetrahydrocannabinol carboxylic acid (THC-COOH). delete The method of claim 1, wherein the liquid phase extraction using the beads is carried out at 10 ± 2 Hz after adding a mixed solvent normal nucleic acid: ethyl acetate (9: 1, v / v) to the sample obtained in the hydrolysis step. Hemp metabolite analysis method, characterized in that the extraction. delete The method according to claim 1, wherein the derivative compound is pentafluoropropionic anhydride (PFPA) / pentafluoropropanol (PFPOH). The method of claim 2, wherein the mass spectrometer uses a tandem mass spectrometer.

Images